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At least 595 records · Page 33Linked to original sources
PubMed1998

The maltose-binding protein as a scaffold for monovalent display of peptides derived from phage libraries.

Random peptide libraries are displayed on filamentous bacteriophage as fusions to either the minor coat protein, pIII, or the major coat protein, pVIII. We have devised a means of isolating the peptide displayed on a phage clone by transferring it to the N-terminus of the maltose-binding protein (MBP) of Escherichia coli encoded by malE. Transfer of a peptide sequence to monomeric MBP eliminates phage-encoded amino acids downstream of the insert peptide as well as avidity effects caused by multivalent display on phage. Peptide:MBP fusions are also easily affinity purified on amylose columns. The pMal-p2 vector was engineered to accept phage DNA encoding pIII- and pVIII-displayed peptides fused to their respective leader sequences. Both types of leader sequence were shown to target the peptide:MBP fusions to the periplasm of E. coli. A streamlined procedure for transferring peptides to MBP was applied to clones that had been isolated from a panel of pVIII-displayed peptide libraries by screening with an HIV-1-specific monoclonal antibody (Ab). By enzyme-linked immunosorbent assay, the Ab bound each of the peptide:MBP fusions and required the presence of a disulfide bridge within each peptide. Some of the peptide:MBP fusions were also analyzed using surface plasmon resonance. Thus, our study shows the value of malE fusion vectors in characterizing phage-displayed peptides.

ATP-Binding Cassette Transporters↗
PubMed2002

[Epitope mapping of hepatitis C virus non-structure protein 5 from a 7 peptide phage library by using immobilized specific monoclonal antibody].

OBJECTIVE: To screen HCV NS5 mimotopes by using monoclonal antibody and phage peptide library. METHODS: By using HCV NS5 monoclonal antibody as selective molecule, a 7 peptide phage library was biopanned and positive clones were selected by ELISA, competition assay and DNA sequencing. RESULTS: Twelve positive clones were chosen for DNA sequencing. From the experiment and sequencing comparison results, one epitope was confirmed as the mimotope of HCV NS5. CONCLUSIONS: HCV mimotope is obtained by phage peptide library screening. The result provides a new approach for HCV therapy and vaccine development.

Amino Acid Sequence↗
PubMed2003

An oriented peptide array library (OPAL) strategy to study protein-protein interactions.

One of the major questions in signal transduction is how the specificities of protein-protein interactions determine the assembly of distinct signaling complexes in response to stimuli. Several peptide library methods have been developed and widely used to study protein-protein interactions. These approaches primarily rely on peptide or DNA sequencing to identify the peptide or consensus motif for binding and may prove too costly or difficult to accommodate high throughput applications. We report here an oriented peptide array library (OPAL) approach that should facilitate high throughput proteomic analysis of protein-protein interactions. OPAL integrates the principles of both the oriented peptide libraries and array technologies. Hundreds of pools of oriented peptide libraries are synthesized as amino acid scan arrays. We demonstrate that these arrays can be used to map the specificities of a variety of interactions, including antibodies, protein domains such Src homology 2 domains, and protein kinases.

Animals↗
PubMedDate not supplied

From consensus sequence to high-affinity ligands: acquisition of signaling protein modulators.

Protein kinases recognize and bind to specific amino acid sequences on their protein substrates. These sequences can be readily identified using combinatorial peptide libraries. Unfortunately, conventional peptide libraries are not designed to identify subtle structural factors that can dramatically enhance enzyme affinity since the "local" diversity associated with these libraries is limited to the 20 standard amino acids. A parallel synthesis strategy was developed that possesses 2 key attributes: moderate size ( approximately 1000 members each), yet high structural diversity (50-fold greater than that of conventional peptide libraries). Due to their small size, these libraries can be synthesized in parallel, which allows each library member to be individually evaluated, and eliminates the requirement for subsequent structural deconvolution. Furthermore, since these libraries possess a high structural diversity focused within narrow spatial windows on the target protein, small regions of the protein can be challenged with a multitude of functionality containing structural differences that vary from subtle to gross.

Combinatorial Chemistry Techniques↗
PubMed2005

Efficient synthesis of a beta-peptide combinatorial library with microwave irradiation.

The predictable relationship between beta-amino acid sequence and folding has inspired several biological applications of beta-peptides. For many such applications, it would be desirable to prepare and screen beta-peptide libraries. However, standard peptide synthesis protocols are not efficient enough to support a library approach for many types of beta-peptides. We recently optimized the solid-phase synthesis of beta-peptides using microwave irradiation, and we have now adapted this approach to synthesis on polystyrene macrobeads. We rapidly prepared a high-quality beta-peptide combinatorial library via a split-and-mix strategy. This library was screened in search of beta-peptide antagonists of the p53-MDM2 protein-protein interaction.

Carbohydrate Sequence↗
PubMed2001

A novel PCNA-binding motif identified by the panning of a random peptide display library.

Proliferating cell nuclear antigen (PCNA) has recently been identified as a target for the binding of proteins involved in DNA replication, DNA repair, and cell cycle control. The interactions between PCNA and a number of these proteins are known to be mediated by a conserved peptide motif. In this study, a random peptide library in which peptide sequences are displayed on the E. coli bacterial flagellin protein was screened for PCNA-binding sequences. Analysis of the retrieved peptide sequences verified the presence of the known PCNA-binding motif. In addition, a second, larger group of peptides containing a different consensus sequence for PCNA binding was discovered. This sequence was found to be present on DNA polymerase delta, and a peptide conforming to this sequence was demonstrated to bind to PCNA. Database search and analysis show that many proteins contain the second consensus sequence. These include proteins that are involved in DNA replication, repair, and cell cycle control. The demonstration of this second PCNA-binding motif may provide a basis for identifying and experimentally testing specific proteins for the structural basis for PCNA binding.

Amino Acid Motifs↗
PubMed1999

Ligands for kappa-opioid and ORL1 receptors identified from a conformationally constrained peptide combinatorial library.

We have screened a synthetic peptide combinatorial library composed of 2 x 10(7) beta-turn-constrained peptides in binding assays on four structurally related receptors, the human opioid receptors mu, delta, and kappa and the opioid receptor-like ORL1. Sixty-six individual peptides were synthesized from the primary screening and tested in the four receptor binding assays. Three peptides composed essentially of unnatural amino acids were found to show high affinity for human kappa-opioid receptor. Investigation of their activity in agonist-promoted stimulation of [(35)S]guanosine 5'-3-O-(thio)triphosphate binding assay revealed that we have identified the first inverse agonist as well as peptidic antagonists for kappa-receptors. To fine-tune the potency and selectivity of these kappa-peptides we replaced their turn-forming template by other turn mimetic molecules. This "turn-scan" process allowed the discovery of compounds with modified selectivity and activity profiles. One peptide displayed comparable affinity and partial agonist activity toward all four receptors. Interestingly, another peptide showed selectivity for the ORL1 receptor and displayed antagonist activity at ORL1 and agonist activity at opioid receptors. In conclusion, we have identified peptides that represent an entirely new class of ligands for opioid and ORL1 receptors and exhibit novel pharmacological activity. This study demonstrates that conformationally constrained peptide combinatorial libraries are a rich source of ligands that are more suitable for the design of nonpeptidal drugs.

Amino Acid Sequence↗
PubMed2005

Bioactive peptides from libraries.

New ligands for a variety of biological targets can be selected from biological or synthetic combinatorial peptide libraries. The use of different libraries to select novel peptides with potential therapeutic applications is reviewed. The possible combination of molecular diversity provided by combinatorial libraries and a rational approach derived from computational modeling is also considered. Advantages and disadvantages of different approaches are compared. Possible strategies to bypass loss of peptide bioactivity in the transition from ligand selection to in vivo use are discussed.

Animals↗
PubMed1998

The role of structure in antibody cross-reactivity between peptides and folded proteins.

Peptides have the potential for targeting vaccines against pre-specified epitopes on folded proteins. When polyclonal antibodies against native proteins are used to screen peptide libraries, most of the peptides isolated align to linear epitopes on the proteins. The mechanism of cross-reactivity is unclear; both structural mimicry by the peptide and induced fit of the epitope may occur. The most effective peptide mimics of protein epitopes are likely to be those that best mimic both the chemistry and the structure of epitopes. Our goal in this work has been to establish a strategy for characterizing epitopes on a folded protein that are candidates for structural mimicry by peptides. We investigated the chemical and structural bases of peptide-protein cross-reactivity using phage-displayed peptide libraries in combination with computational structural analysis. Polyclonal antibodies against the well-characterized antigens, hen eggwhite lysozyme and worm myohemerythrin, were used to screen a panel of phage-displayed peptide libraries. Most of the selected peptide sequences aligned to linear epitopes on the corresponding protein; the critical binding sequence of each epitope was revealed from these alignments. The structures of the critical sequences as they occur in other non-homologous proteins were analyzed using the Sequery and Superpositional Structural Assignment computer programs. These allowed us to evaluate the extent of conformational preference inherent in each sequence independent of its protein context, and thus to predict the peptides most likely to have structural preferences that match their protein epitopes. Evidence for sequences having a clear structural bias emerged for several epitopes, and synthetic peptides representing three of these epitopes bound antibody with sub-micromolar affinities. The strong preference for a type II beta-turn predicted for one peptide was confirmed by NMR and circular dichroism analyses. Our strategy for identifying conformationally biased epitope sequences provides a new approach to the design of epitope-targeted, peptide-based vaccines.

Amino Acid Sequence↗
PubMed2006

Identification of peptide mimetics of xenoreactive alpha-Gal antigenic epitope by phage display.

The prevention of hyperacute rejection (HAR) triggered by interaction between the human natural antibody and xenoreactive antigenic epitope (Gal-alpha1, 3Gal) present on pig cells is the key to success in pig-to-human xenotransplantation. The phage display technology offers an effective strategy for screening peptides which can interact with the anti-Gal antibody to block alpha-Gal antigen binding site. Two peptide libraries, linear 7 peptide library and C7C library, were panned on the anti-B monoclonal antibody which has the characteristic of binding to the alpha-Gal antigenic epitope. After four rounds of panning, 22 positive phage clones were selected. Highly homologous sequence PT and STL existed among these selected peptides. Stachyose competitive ELISAs revealed that these peptides specifically bound to alpha-Gal antigen binding site. Eight peptide mimics of alpha-Gal antigenic epitope could inhibit the agglutination of pig red blood cells mediated by human sera in a dose-dependent manner. These results demonstrated that the selected peptides can mimic the conformational structure of alpha-Gal antigenic epitope and have the therapeutic potential in xenotransplantation.

Agglutination↗
PubMed2002

Parallel synthesis of PNA-peptide conjugate libraries.

An optimized semi-automatic protocol for parallel synthesis of up to 96 peptide nucleic acids (PNA) or PNA-peptide conjugates using Boc-protection strategy has been developed using a robotic system. The approach is illustrated by synthesizing PNA and PNA-peptide libraries varying between 15 and 27 amino acid units. The peptides (NLS (nuclear localization signal) or Tat-peptide) were attached to N-terminus of the PNA. The method was found to be far superior to that based on the SPOT/Fmoc protocol by which PNA oligomers are synthesized on a modified cellulose membrane. On a 0.5 micromole scale the method typically yielded 2 mg product of 90% purity by HPLC/MALDI-TOF analysis. This approach is suitable for screening of a large number of PNA and/or peptide sequences for biochemical and biological studies.

Amino Acid Sequence↗
PubMed1999

A rheumatoid factor specific mimotope identified by a peptide display library.

We screened a 10 amino acid peptide display library in filamentous phage with B'20, a monoclonal high affinity IgM rheumatoid factor (RF) expressing the VkIIIa-dependent 4C9 idiotype. Using direct and indirect selection techniques, 12 B'20 reactive peptides were identified, 9 of which belonged to one of two motifs. Binding of B'20 to phage-bearing peptides was inhibited by both IgG and 4C9 antiidiotype. Synthetic peptides corresponding to the two motifs inhibited the Fc binding of a low avidity IgA B'20 construct. Purified IgM from 6/8 RF-positive RA patients and 8/11 monoclonal RFs with VkIII-encoded light chains bound to the phage, whereas none of the four monoclonal RFs with VkI or VkII encoded light chains bound. Phage binding appeared to be RF specific. Three 4C9 positive/RF negative cell lines from RA patients did not bind to phage nor did three B'20 mutants that had lost RF specificity, whereas two mutants that retained RF specificity also retained phage binding. We propose that there is a common epitope(s) recognized by VkIII encoded RFs that is mimicked by the structure of these peptides. Such mimotopes might be exploited to design novel agents that interfere with autoantibody binding.

Antibodies, Monoclonal↗
PubMed1996

Novel alpha-glucosidase inhibitors identified using multiple cyclic peptide combinatorial libraries.

Twenty-six cyclic synthetic peptide combinatorial libraries (disulfides and lactams) of varying size and composition, representing 6.8 x 10(3) to 4.7 x 10(7) individual peptides, were synthesized along with their respective linear analogs. One of the hexapeptide lactam libraries (cyclo[xXxXxN]) was found to have significant alpha-glucosidase inhibitory activity. This library was carried through an iterative process of synthesis and screening, during which all of the five mixture positions (x and X) were successively defined. As the result of this process, potent and selective alpha-glucosidase inhibitors were identified.

Amino Acid Sequence↗
PubMed2005

Anti-flt1 peptide, a vascular endothelial growth factor receptor 1-specific hexapeptide, inhibits tumor growth and metastasis.

PURPOSE: The purpose of this study was to develop antagonists specific for the vascular endothelial growth factor receptor 1 (VEGFR1) and to investigate the effects of the antagonists on the VEGF-induced endothelial cell functions and tumor progression. EXPERIMENTAL DESIGN: Hexapeptides that inhibit binding of VEGFR1 and VEGF were identified through screening of synthetic peptide library. A selected peptide, anti-Flt1, was investigated for binding specificity with various receptors and ligand peptides. Effects of the peptide on proliferation, cell migration, and fibrin gel-based angiogenesis of endothelial cells were also investigated. The activity of anti-Flt1, in vivo, was evaluated for inhibition of tumor growth and metastasis in VEGF-secreting cancer cell-implanted mice by s.c. injections of the peptide. RESULTS: Here, we report on a short peptide that binds to VEGFR1 and prevents binding of VEGF. A hexapeptide, anti-Flt1 (Gly-Asn-Gln-Trp-Phe-Ile or GNQWFI), was identified from peptide libraries. The anti-Flt1 peptide shows specificity toward binding to VEGFR1 and it inhibits binding of VEGF, placental growth factor (PlGF), and VEGF/PlGF heterodimer to VEGFR1. This peptide does not inhibit the proliferation of endothelial cells induced by VEGF and VEGF/PlGF heterodimer but it effectively blocks VEGF-induced migration of endothelial cells and their capacity to form capillary-like structures on fibrin gel-based in vitro angiogenesis system. Furthermore, growth and metastasis of VEGF-secreting tumor cells were also significantly inhibited by s.c. injections of anti-Flt1 peptide in nude mice. Accordingly, VEGF-induced migration and capillary formation are mediated through VEGFR1, and these processes may play an important role in the growth and metastasis of VEGF-secreting tumors. CONCLUSIONS: We show that a peptide (anti-Flt1) specific for VEGFR1 inhibits growth and metastasis of tumor that secretes VEGF. The effects on endothelial cell functions, in vitro, indicate that the anticancer activity of anti-Flt1 peptide with reduced blood vessel density could also be due to the blocking of VEGFR1-mediated endothelial cell migration and tube formation. Although the effects of anti-Flt1 peptide still remain to be further characterized, the receptor 1-specific peptide antagonist, anti-Flt1, has potential as a therapeutic agent for various angiogenesis-related diseases, especially cancer.

Amino Acid Sequence↗
PubMed1995

Rapid identification of highly active and selective substrates for stromelysin and matrilysin using bacteriophage peptide display libraries.

The discovery of useful peptide substrates for proteases that recognize many amino acids in their active sites is often a slow process due to the lack of initial substrate data and the expense of analyzing large numbers of peptide substrates. To overcome these obstacles, we have made use of bacteriophage peptide display libraries. We prepared a random hexamer library in the fd-derived vector fAFF-1 and included a "tether" sequence that could be recognized by monoclonal antibodies. We chose the matrix metalloproteinases stromelysin and matrilysin as the targets for our studies, as they are known to require at least 6 amino acids in a peptide substrate for cleavage. The phage library was treated in solution with protease and cleaved phage separated from uncleaved phage using a mixture of tether-binding monoclonal antibodies and Protein A-bearing cells followed by precipitation. Clones were screened by the use of a rapid screening assay that identified phage encoding peptide sequences susceptible to cleavage by the enzymes. The nucleotide sequence of the random hexamer region of 43 such clones was determined for stromelysin and 23 for matrilysin. Synthetic peptides were prepared whose sequences were based on some of the positive clones, as well as consensus sequences built from the positive clones. Many of the peptides have kcat/KM values as good or better than those of previously reported substrates, and in fact, we were able to produce stromelysin and matrilysin substrates that are both the most active and smallest reported to date. In addition, the phage data predicted selectivity in the P2 and P'1 positions of the two enzymes that were supported by the kinetic analysis of the peptides. This work demonstrates that the phage selection techniques enable the rapid identification of highly active and selective protease substrates without making any a priori assumptions about the specificity or the "physiological substrate" of the protease under study.

Amino Acid Sequence↗
PubMed2001

Automatic Edman microsequencing of peptides containing multiple unnatural amino acids.

It is now routine using automatic Edman microsequencing to determine the primary structure of peptides or proteins containing natural amino acids; however, a deficiency in the ability to readily sequence peptides containing unnatural amino acids remains. With the advent of synthetic peptide chemistry, combinatorial chemistry, and the large number of commercially available unnatural amino acids, there is a need for efficient and accurate structure determination of short peptides containing many unnatural amino acids. In this study, 35 commercially available alpha-unnatural amino acids were selected to determine their elution profile on an ABI protein sequencer. Using a slightly modified gradient program, 19 of these 35 PTH amino acids can be readily resolved and distinguished from common PTH amino acids at low picomole levels. These unnatural amino acids in conjunction with the 20 natural amino acids can be used as building blocks to construct peptide libraries, and peptide beads isolated from these libraries can be readily microsequenced. To demonstrate this, we synthesized a simple tripeptide "one-bead one-compound" combinatorial library containing 14 unnatural and 19 natural amino acids and screened this library for streptavidin-binding ligands. Microsequencing of the isolated peptide-beads revealed the novel motif Bpa-Phe(4-X)-Aib, wherein X = H, OH, and CH3.

Amino Acids↗
PubMed1997

Synthetic peptide combinatorial libraries: a method for the identification of bioactive peptides against phytopathogenic fungi.

Synthetic combinatorial libraries were evaluated with an iterative process to identify a hexapeptide with broadspectrum activity against selected phytopathogenic fungi. A D-amino acid hexapeptide (FRLKFH) and pentapeptide (FRLHF) exhibited activity against Fusarium oxysporum f. sp. lycopersici, Rhizoctonia solani (anastomosis group 1), Ceratocystis fagacearum, and Pythium ultimum. The peptides showed no hemolytic or mutagenic activity. Fluorescent microscopy studies with a membrane impermeant dye indicated that fungal cytoplasmic membranes were compromised rapidly and that the nuclear membrane was also affected.

Amino Acid Sequence↗
PubMed2004

CIS display: In vitro selection of peptides from libraries of protein-DNA complexes.

We describe the development of an in vitro library selection system (CIS display) that exploits the ability of a DNA replication initiator protein (RepA) to bind exclusively to the template DNA from which it has been expressed, a property called cis-activity. A diverse peptide library is created by ligation of DNA fragments of random sequence to a DNA fragment that encodes RepA. After in vitro transcription and translation, a pool of protein-DNA complexes is formed where each protein is stably associated with the DNA that encodes it. These complexes are amenable to the affinity selection of ligands to targets of interest. Here we show that RepA is a highly faithful cis-acting DNA-binding protein and demonstrate that libraries encoding >10(12) random 18-mer peptides can be constructed and used to isolate peptides that bind specifically to disparate targets. The use of DNA to encode the displayed peptides offers advantages over in vitro peptide display systems that use mRNA.

Amino Acid Sequence↗